Monolithic Capillary Column Based Glycoproteomic Reactor for High-Sensitive Analysis of N‑Glycoproteome Jing Liu Fangjun Wang Hui Lin Jun Zhu Yangyang Bian Kai Cheng Hanfa Zou 10.1021/ac400315n.s003 https://acs.figshare.com/articles/journal_contribution/Monolithic_Capillary_Column_Based_Glycoproteomic_Reactor_for_High_Sensitive_Analysis_of_N_Glycoproteome/2437534 Despite the importance of protein N-glycosylation in a series of biological processes, in-depth characterization of protein glycosylation is still a challenge due to the high complexity of biological samples and the lacking of highly sensitive detection technologies. We developed a monolithic capillary column based glycoproteomic reactor enabling high-sensitive mapping of N-glycosylation sites from minute amounts of sample. Unlike the conventional proteomic reactors with only strong-cation exchange or hydrophilic-interaction chromatography columns, this novel glycoproteomic reactor was composed of an 8 cm long C12 hydrophobic monolithic capillary column for protein digestion and a 6 cm long organic–silica hybrid hydrophilic monolithic capillary column for glycopeptides enrichment and deglycosylation, which could complete whole-sample preparation including protein purification/desalting, tryptic digestion, enrichment, and deglycosylation of glycopeptides within about 3 h. The developed reactor exhibited high detection sensitivity in mapping of N-glycosylation sites by detection limit of horseradish peroxidase as low as 2.5 fmol. This reactor also demonstrated the ability in complex sample analysis, and in total, 486 unique N-glycosylation sites were reliably mapped in three replicate analyses of a protein sample extracted from ∼10<sup>4</sup> HeLa cells. 2013-03-05 00:00:00 Monolithic Capillary Column capillary column detection glycopeptide cm novel glycoproteomic reactor deglycosylation site sample protein mapping enrichment